AQP3-mediated H₂O₂ Transport Drives Macrophage M1 Polarization and Cervical Matrix Remodeling in Cervical Insufficiency†.

Background Colon adenocarcinoma (COAD) is a leading cause of cancer mortality, with Aquaporin 9 (AQP9) implicated in its progression. M2 macrophages in the tumor microenvironment (TME) promote cancer metastasis, but the role of AQP9 on M2 macrophages remains unelucidated. Research design and methods Using COAD cell lines, AQP9 expression was analyzed via RT-qPCR and Western blot (WB). Hypoxic conditions were simulated to assess HIF-1α and AQP9 interactions through ChIP and dual-luciferase assays. AQP9 knockdown effects on proliferation/migration were tested via colony formation and wound healing. M2 macrophage polarization and CD8+ T cell cytotoxicity were evaluated using flow cytometry, ELISA, and IHC in co-culture systems. Results AQP9 was upregulated in COAD and correlated with poor prognosis. After AQP9 in COAD cells was knocked down, the abilities of tumor cells to migrate and proliferate were dampened. Hypoxia upregulated HIF-1α, which transcriptionally activated AQP9. Knocking down AQP9 repressed the M2 polarization of macrophages, thereby reinforcing the cytotoxicity of CD8+ T cells. No adverse events were reported in vitro. Conclusion AQP9 promotes COAD progression by driving HIF-1α-mediated M2 polarization, impairing CD8+ T cell function. Key limitations include the lack of in vivo validation and clinical cohort analysis.

Cross-communication between cancer cells and macrophages within the tumor microenvironment fulfills the critical roles in the progression of cancers, including hepatocellular carcinoma (HCC). Ligustilide exerts anti-inflammation, anti-injury, and anti-tumor pleiotropic pharmacological functions. Nevertheless, its roles in HCC cells and tumor microenvironment remain elusive. In the current study, ligustilide dramatically restrained HCC cell viability and migration but had little cytotoxicity to normal hepatocytes. Importantly, ligustilide antagonized HCC cell co-culture-induced macrophage recruitment and M2 polarization by enhancing the percentage of CD14+CD206+ cells and macrophage M2 markers (CD163, Arg1, CD206, CCL22, IL-10, and TGF-β). Mechanistically, ligustilide repressed yes-associatedprotein (YAP) activation by reducing nuclear translocation, protein expression, transcriptional regulatory activity of YAP, and increasing p-YAP levels. Noticeably, blocking the YAP offset the suppressive effects of ligustilide on macrophage recruitment and M2 polarization evoked by HCC cells. Moreover, the release of interleukin-6 (IL-6) was mitigated by ligustilide in a YAP-dependent manner in HCC cells, concomitant with inhibition of IL-6R/STAT3 signaling activation. Of interest, interdicting the IL-6 aggravated ligustilide-mediated suppression in HCC-induced macrophage recruitment and M2 polarization; whereas exogenous IL-6 treatment reversed the above effects. Additionally, blockage of IL-6R signaling also overturned IL-6-induced macrophage recruitment and M2 phenotype. Consequently, these findings support a notion that ligustilide not only restrains HCC cell malignancy but also antagonizes HCC cell-evoked macrophage recruitment and M2 polarization by inhibiting YAP/IL-6 release-induced activation of the IL-6 receptor/signal transducer and activator of transcription 3 (IL-6R/STAT3) signaling. Thus, ligustilide may be a promising therapeutic agent to fight HCC by regulating cancer cells and cross-talk between tumor cells and macrophages in tumor microenvironment.

Breast cancer is the leading cause of death in female cancer patients due to the lack of effective treatment for metastasis. Macrophages are the most abundant immune cells in the primary and metastatic tumors, and contribute to tumor initiation, progression, and metastasis. Emodin has been found to exert anti-tumor effects through promoting cell cycle arrest and apoptosis, and inhibiting angiogenesis, but its effects on tumor-associated macrophages during cancer metastasis have not been investigated. Mice inoculated with 4T1 or EO771 breast cancer cells orthotopically were treated with Emodin after the primary tumors reached 200 mm3 in size. Primary tumor growth, lung metastasis, and macrophage infiltration in the lungs were analyzed. In vitro experiments were performed to examine the effects of Emodin on macrophage migration and M2 polarization, and the underlying mechanisms. Emodin significantly suppressed breast cancer lung metastasis in both orthotopic mouse models without apparent effects on primary tumors. Reduced infiltration of F4/80+ macrophages and Ym1+ M2 macrophages in lungs was observed in Emodin-treated mice. In vitro experiments demonstrated that Emodin decreased the migration of macrophages toward tumor cell-conditioned medium (TCM) and inhibited macrophage M2 polarization induced by TCM. Mechanistically, Emodin suppressed STAT6 phosphorylation and C/EBPβ expression, two crucial signaling events in macrophage M2 polarization, in macrophages treated with IL-4 or TCM. Taken together, our study, for the first time, demonstrated that Emodin suppressed pulmonary metastasis of breast cancer probably through inhibiting macrophage recruitment and M2 polarization in the lungs by reducing STAT6 phosphorylation and C/EBPβ expression.

Lipopolysaccharide (LPS)-induced acute kidney injury (AKI) is associated with an abnormal immune response. Accumulating evidence has demonstrated that aquaporin 1 (AQP1) prevents kidney tissue injury in LPS-induced AKI by mediating immune response. However, the underlying mechanisms remain obscure. Macrophages as immune cells with multiple phenotypes are important mediators in tissue homeostasis and host defense. We propose that macrophage polarization is implicated in AQP1-mediated immune response. Herein we established sepsis-induced AKI model rats through intraperitoneal injection of LPS into Wistar rats to reveal immune mechanism of damage. We also used LPS-induced mouse RAW264.7 cells to elucidate the molecular mechanism of macropage polarization. Histopathology showed that renal tubular epithelial cells in the model group were swollen, inflammatory exudation was obvious and the inflammatory factors, interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α) were increased. Western blotting showed PI3K was upregulated in the model group. Serum creatinine and urea nitrogen increased after LPS injection. Renal AQP1 mRNA is downregulated and serum AQP1 protein increased first and then decreased in LPS-induced AKI rats. M2 macrophage markers (Arg-1, CD206) were increased in repair stage. In addition, treatment of murine macrophages (RAW264.7) with AQP1 siRNA resulted in decreased PI3K activation and M2 polarization, but increased IL-6 and TNF-α. Moreover, inhibiting PI3K with wortmannin imitated the results of AQP1 silencing. Macrophage M2 polarization is likely the cellular mechanism underlying the anti-AKI property of AQP1, and PI3K activation is involved in the AQP1-induced M2 phenotype switch.

Inhibition of Kv1.3 channel restrains macrophage M2 polarization and ameliorates renal fibrosis via regulating STAT6 phosphorylation.

BackgroundSphingosine kinase 1 (SphK1) has distinct roles in the activation of Kupffer cells (KCs) and hepatic stellate cells (HSCs) in liver fibrosis. This study aims to examine the role of...

Aquaporin 3 (AQP3), which is mostly expressed in pulmonary epithelial cells, was linked to lung adenocarcinoma (LUAD). However, the underlying functions and mechanisms of AQP3 in the tumor microenvironment (TME) of LUAD have not been elucidated. Single-cell RNA sequencing (scRNA-seq) was used to study the composition, lineage, and functional states of TME-infiltrating immune cells and discover AQP3-expressing subpopulations in five LUAD patients. Then the identifications of its function on TME were examined in vitro and in vivo. AQP3 was associated with TNM stages and lymph node metastasis of LUAD patients. We classified inter- and intra-tumor diversity of LUAD into twelve subpopulations using scRNA-seq analyses. The analysis showed AQP3 was mainly enriched in subpopulations of M2 macrophages. Importantly, mechanistic investigations indicated that AQP3 promoted M2 macrophage polarization by the PPAR-γ/NF-κB axis, which affected tumor growth and migration via modulating IL-6 production. Mixed subcutaneous transplanted tumor mice and Aqp3 knockout mice models were further utilized, and revealed that AQP3 played a critical role in mediating M2 macrophage polarization, modulating glucose metabolism in tumors, and regulating both upstream and downstream pathways. Overall, our study demonstrated that AQP3 could regulate the proliferation, migration, and glycometabolism of tumor cells by modulating M2 macrophages polarization through the PPAR-γ/NF-κB axis and IL-6/IL-6R signaling pathway, providing new insight into the early detection and potential therapeutic target of LUAD.

Objective To explore the mechanism of ubiquitin specific peptidase 21 (USP21) increasing the stability of forkhead box protein M1 (FOXM1) and promoting M2 polarization of macrophages in endometriosis (EM). Methods Eutopic endometrial stromal cells (EESC) collected from patients and normal endometrial stromal cells (NESC) from routine health examiners were cultured in vitro, and the expression levels of USP21 and FOXM1 were detected using RT-qPCR and Western blot. EESCs were co-cultured with macrophages. M1 polarization markers of interleukin 6 (IL-6) and CXC chemokine ligand 10 (CXCL10) and M2 polarization markers of CD206 and fibronectin 1 (FN1) were tested using RT-qPCR. M2 marker CD206 was further detected by flow cytometry. IL-6, tumor necrosis factor-alpha (TNF-α), IL-10, and transforming growth factor-beta (TGF-β) levels in cell supernatant were detected by ELISA. Co-immunoprecipitation was used to assess the interaction between USP21 and FOXM1, and the ubiquitination level of FOXM1. FOXM1 protein stability was detected through cycloheximide (CHX) assay. Results USP21 and FOXM1 expression levels in the EESC group were significantly increased compared with those in the NESC group; compared with the NESC + M0 group, the EESC + M0 group showed no significant difference in the expression of M1 polarization markers (IL-6 and CXCL10), but increased expression of M2 polarization markers (CD206 and FN1), along with notably increased number of M2 macrophages; there was no significant difference in IL-6 and TNF-α levels, but increased levels of IL-10 and TGF-β in the cell supernatant. The above findings indicated that the deubiquitinase USP21 was highly expressed in EM, promoting M2 polarization of macrophages. Knocking down USP21 or FOXM1 can inhibit M2 polarization of EM macrophages. USP21 interacted with FOXM1 in EESC, leading to a decrease in FOXM1 ubiquitination level and an increase in FOXM1 protein stability. Overexpression of FOXM1 reversed the inhibitory effect of knocking down USP21 on M2 polarization of EM macrophages. Conclusion The deubiquitinase USP21 interacts with FOXM1 to increase the stability of FOXM1 and promote M2 polarization of EM macrophages.

The important enteropathogen Salmonella can cause lethal systemic infection via survival and replication in host macrophages. Lactate represents an abundant intracellular metabolite during bacterial infection, which can also induce macrophage M2 polarization. In this study, we found that macrophage-derived lactate promotes the intracellular replication and systemic infection of Salmonella. During Salmonella infection, lactate via the Salmonella type III secretion system effector SteE promotes macrophage M2 polarization, and the induction of macrophage M2 polarization by lactate is responsible for lactate-mediated Salmonella growth promotion. This study highlights the complex interactions between Salmonella and macrophages and provides an additional perspective on host-pathogen crosstalk at the metabolic interface.

SLC16A3 is considered to affect the malignant progression of lung adenocarcinoma (LUAD), but its mechanism remains elusive. Lactate secretion can facilitate the M2 polarization of macrophages, which are essential components of the tumor immune microenvironment (TIME). Based on the Cancer Genome Atlas (TCGA) database, differential expression analysis of SLC16A3 in LUAD was undertaken and the Pearson correlation analysis was on SLC16A3 and targets of M2 macrophages. Pathway enrichment analysis on SLC16A3 was achieved by utilizing the gene set enrichment analysis (GSEA). The expression of SLC16A3 in cells was examined by qPCR and Western blot (WB). The levels of glycolysis marker proteins in cells were tested by WB. The Glucose test kit, lactate test kit, Seahorse energy metabolism analyzer, and pHrodo™ Green AM intracellular indicator reagent kit were applied in assessing cellular glycolysis levels. CCK-8, scratch assay, Transwell assay, and flow cytometry were conducted to evaluate the malignant phenotype and apoptosis level of cancer cells. Flow cytometry and Enzyme-linked immunosorbent assay (ELISA) were utilized to assess the polarization of macrophages. Finally, a mouse model of allograft tumors was created, and the effects of SLC16A3 on glycolysis and M2 polarization of macrophages in vivo were evaluated by tracking tumor growth and detecting related protein distribution through Immunohistochemistry. SLC16A3 was greatly upregulated in LUAD. Knocking down SLC16A3 remarkably repressed the malignant phenotype of LUAD cells and reinforced apoptosis. The results derived from GSEA manifested that SLC16A3 had a higher enrichment in the glycolysis pathway. SLC16A3 positively modulated the extracellular and intracellular levels of lactate and glycolysis. Pearson correlation analysis uncovered a positive linkage between SLC16A3 and M2 macrophage markers. According to the rescue experiment, glycolysis inhibitors were observed to greatly reduce the enhancement in M2 polarization of macrophages caused by overexpression of SLC16A3. The final mouse experiment demonstrated that SLC16A3 boosted tumor growth in vivo and enhanced tumor glycolysis level and M2 macrophage infiltration in the TIME. SLC16A3 in LUAD modulates the glycolysis pathway to facilitate M2 polarization of macrophages.

1-Ethoxycarbonyl-beta-carboline inhibits the M2 polarization of tumor-associated macrophages: A study based on network pharmacology and molecular docking analyses

The chemokine CCL1 facilitates pulmonary fibrosis by promoting macrophage migration and M2 polarization

Berberine (BBR), an isoquinoline alkaloid extracted from Coptis chinensis, is clinically used to treat chronic colitis, diabetes, and other diseases. Although BBR has antitumor effects, it is unclear whether it can inhibit hepatocellular carcinoma (HCC) by modulating the tumor inflammatory microenvironment. In this study, we demonstrated that BBR inhibits HCC development in mice by suppressing the M2 polarization of macrophages. Using an H22 tumor-bearing xenograft mouse model, we found that BBR significantly inhibited H22 tumor growth. Analysis of scRNA-seq results revealed reduced M2 macrophage infiltration and polarization in BBR-treated HCC tissues. Pharmacodynamic studies showed that BBR treatment markedly increased CD8+ T cell infiltration and attenuated M2 polarization. In vitro, BBR suppressed IL-4 or tumor cell supernatant-induced M2 polarization, as evidenced by decreased expression of M2 polarization marker genes (Arg1, Retnla, etc.) and reduced JAK1/STAT6 phosphorylation levels. Molecular docking and protein stability assays revealed that BBR directly binds to JAK1’s FERM domain, stabilizing it. Combination therapy with BBR and anti-PD-L1 antibody synergistically inhibited H22 tumor growth. These findings suggest that BBR can reduce the M2 polarization of tumor-associated macrophages (TAMs) by targeting the IL-4-JAK1-STAT6 axis, and combining with anti-PD-L1 antibody may represent a promising therapeutic strategy to enhance BBR’s antitumor efficacy.

Lung adenocarcinoma (LUAD) is one of the frequent subtypes of lung cancer, featuring high rates of incidence and mortality. Matrix metalloproteinase 14 (MMP14) is known as a regulator in multiple cancers, whereas its upstream molecular mechanism remains to be investigated. This study aims to reveal the upstream molecular mechanism of MMP14 in LUSC progression. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blot were conducted to examine the levels of MMP14 mRNA and protein in LUAD cells, respectively. Cell counting kit-8 (CCK-8), transwell assay and wound healing assay were implemented to unveil LUAD cell proliferation, migration and invasion after indicated transfections. Flow cytometry analysis was applied to evaluate macrophage polarization. Mechanism experiments such as western blot, co-immunoprecipitation (Co-IP), RNA pulldown assay, luciferase reporter assay and RNA-binding protein immunoprecipitation (RIP) assay were used to explore relevant molecular mechanisms. MMP14 facilitated LUAD cell proliferation, invasion and migration. MMP14 is the target gene of miR-1287-5p. Circ-ADRM1 upregulates MMP14 expression through sponging miR-1287-5p. Circ-ADRM1 recruits USP12 to impede the ubiquitination of MMP14 protein, thereby enhancing the stability of MMP14 protein. LUAD-derived exosomes induced macrophage M2 polarization by delivering circ-ADRM1. Circ-ADRM1 promotes LUAD cell proliferation, invasion and migration through upregulating MMP14. Additionally, circ-ADRM1 induces macrophage M2 polarization in an exosome-dependent manner.

Sepsis-associated acute kidney injury (AKI) has high morbidity and mortality, but without cause-specific treatment. Erythropoietin derived Helix B surface peptide (HBSP) alleviates AKI, whereas its underlying mechanisms remain to be further explored. Here, the effects of HBSP on pyroptosis, apoptosis, macrophage polarization and repair were investigated in lipopolysaccharide (LPS)-induced AKI mouse model and cultured kidney epithelial cells. Systemic inflammation, compromised renal function and histology were demonstrated in LPS-treated mice, with upregulated pyroptotic and apoptotic key proteins in the kidneys including GSDMD-N, cleaved IL-1β, IL-18 and caspase-3. These proteins were localized in tubular areas and colocalized with aquaporin-1 (AQP1), with increased F4/80+ M1 macrophages. However, HBSP mitigated pyroptosis, apoptosis and inflammation, and promoted macrophage M2 polarization. In addition, HMGB1 and erythropoietin receptor (EPOR) were increased by LPS and decreased by HBSP, both of which were positively correlated with pyroptotic and apoptotic proteins. Moreover, HBSP reduced TNF-α and IL-6 mRNA levels, as well as pyroptosis and apoptosis in LPS-stimulated TCMK-1 cells. In conclusion, HBSP inhibited tubular pyroptosis and apoptosis, EPOR expression, promoted macrophage M2 polarization, and protected against LPS-induced AKI. These findings provide new mechanistic insights into the renoprotection of HBSP, and facilitate its potential for clinical applications and therapeutic strategies in sepsis-associated AKI.